The transport of macromolecules between the nucleus and the cytoplasm is an essential activity of all eukaryotic cells. We are using the c-Rel transcription factor and its inhibitor, IkappaBalpha, as a model system to understand how the directional transport of proteins between the nucleus and the cytoplasm can provide an effective mechanism for regulation of gene expression. The IkappaBalpha protein can both inhibit nuclear import of c-Rel and direct the export of c-Rel from the nucleus. This nuclear shuttling property of IkappaBalpha enables tight control over the net nuclear abundance of c-Rel. The first hypothesis to be addressed in this proposal is that the nuclear shuttling property of IkappaBalpha is the net sum of nuclear import, nuclear retention, and nuclear export. The amino acids in IkappaBalpha that are responsible for each of these functions will be defined using both in vitro and in vivo assays. Trans-acting factors that participate in nuclear import of IkappaBalpha will be identified and novel mechanistic features of IkappaBalpha nuclear import pathway will be defined. The second hypothesis to be tested in this proposal is that IkappaBalpha controls the nuclear abundance of c-Rel through both inhibition of nuclear import and through nuclear export. The ability of IkappaBalpha to inhibit nuclear import of c-Rel will be contrasted to the inability of IkappaBalpha to inhibit nuclear import of the v-Rel oncoprotein. The function of IkappaBalpha as an adaptor protein that bridges c-Rel with the export receptor, Crm1, will be examined. The ability of Crm1 to discriminate between free IkappaBalpha and c-Rel-associated IkappaBalpha will be determined. An innovative in vitro nuclear export assay will be developed and used to understand how nuclear export is terminated on the cytoplasmic face of the nuclear pore complex. The fate of the c-Rel: IkappaBalpha complex following termination of nuclear export will also be determined.